Dengue virus is a causative agent of dengue fever and dengue hemorrhagic fever, which are widespread in several tropical countries around the world. Transmission of dengue virus among humans occurred through mosquito biting activity; infected human serve principally as the reservoirs for transmission in the urban setting. Among viral isolates obtained from infected humans and mosquitoes, four serologically defined types (serotypes) of dengue virus are known, all of which can cause potentially fatal dengue hemorrhagic fever (D.S. Burke and T. P. Monath (2001) Flaviviruses, P. 1043-1125, in D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman and S. E. Strauss (ed.), Fields Virology, 4th ed. Lippincott Williams & Wilkins, Philadelphia, Pa.). In human, primary infection by dengue virus of any serotype generally causes milder diseases and induces immunity only against the infecting serotype. As cross protective immunity is short-lived, secondary infection by other dengue serotypes is common in countries where two or more serotypes co-circulate. It is well accepted that secondary infection increases the risk of developing dengue hemorrhagic fever. Effective prevention of dengue hemorrhagic fever will require a vaccine, which can induce protective immunity against all four serotypes of dengue virus.
Dengue virus is a member of the Genus Flavivirus in the Family Flaviviridae. The virion is spherical in shape with the diameter of about 50 nm. The outer part of the virion (envelope) consists of lipid bilayer and two different glycoproteins, E and prM/M (B. D. Lindenbach and C. M. Rice. (2001) p. 991-1041 in D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman and S. E. Strauss (ed.), Fields Virology, 4th ed. Lippincott Williams & Wilkins, Philadelphia, Pa.). The core is composed of another protein, C, in association with the single-stranded RNA genome. In each virion, one molecule of about 10.7 kb long RNA genome is present; it encodes three structural proteins and seven non-structural proteins, which are required for virus multiplication inside the infected cells, but are not components of virion. The organization of the genome is as follows: 5′ cap-5′ untranslated region-C-prM/M-E-NS-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3′ untranslated region-3′ end. This genome organization is common to all flaviviruses.
Following the assembly of virion in the endoplasmic reticulum, the envelope of immature virions, consisting of only prM and E proteins, is further modified by glycosylating enzymes in the Golgi apparatus and by the proteolytic enzyme, furin, in the trans-Golgi network. Specifically, when immature virion is exported along the secretory pathway to the extracellular milieu, furin cleaves the protein prM internally at the pr-M junction, generating virion-associated M protein and soluble pr peptide, which no longer associate with the virion (K. Stadler, et al. (1997) J. Virol. 71:8475-8481). Cleavage of prM is absolutely necessary for the ability of the mature, extracellular virion of flavivirus to initiate productive infection of the host cell (S. Elshuber, et al. (2003) J. Gen. Virol. 84:183-191). However, cleavage of prM can be incomplete and extracellular virions of several flaviviruses were known to contain varying amount of prM protein. The significance of the remaining prM on the envelope is not yet known.
Recent structural data of the immature particles of dengue virus reveal that the prM proteins associate with the E proteins as sets of prM-E heterodimer (Y. Zhang, et al. (2003) EMBO J. 22:2604-13). The prM proteins project out of the surface whereas the E proteins lie flat and parallel to the lipid bilayer of the envelope. The protruding portion of the prM proteins covers the tip of the E protein that is responsible for fusing activity of E protein. The structure of a preparation of the mature dengue virion in which all prM proteins are cut is quite different from the immature virion including the formation of E-E homodimers (R. J. JKuhn, et al. (2002) Cell, 108:717-25), indicating that cleavage of prM by furin leads to a significant rearrangement of the envelope proteins. Such rearrangement must be important for generating infectious viral particles.
It has been repeatedly observed that the extracellular virions of dengue virus usually contain remaining prM protein on their envelope (R. Anderson, et al. (1997) J. Virol. 71: 4226-4232; R. T. He, et al.(1995) J. Med. Virol. 45: 451-461; E. A. Henchal et al. (1985) Am. J. Trop. Med. Hyg. 34: 162-169; J. M. Murray et al. (1993) J. Gen. Virol. 74: 175-182; V. B. Randolph et al. (1990) Virology 174: 450-458; J. T. Roehrig et al. (1998) Jamaica. Virology 246: 317-328; S. Wang et al. (1999) J. Virol. 73: 2547-2551). The observations were made in dengue virus prepared from a number of cell lines of both mosquito and mammalian origins in several laboratories. These dengue virus preparations are infectious and able to generate new rounds of infection efficiently.
When compared with other flaviviruses in which the prM protein is completely cleaved, an incomplete cleavage of prM in dengue virus coincides with a lower number of positively charged amino acids and also the presence of two negatively charged amino acids within the 13-amino acid, pr-M junction proximal sequence, which extends beyond the P6 and P4 boundary previously known to affect cleavage of target protein by furin (G. Thomas (2002) Nat. Rev. Mol. Cell. Biol. 3: 753-766; K. Nakayama (1997) Biochem. J. 327: 625-635; A. Zhou et al. (1999) J. Biol. Chem. 274: 20745-20748). Thus, one of the distinctive features of dengue virus is the conservation of the pr-M cleavage junction sequence, which allows only partial cleavage by host cell-derived furin. Alterations of dengue pr-M cleavage junction may modify the structural characteristics of the virion and the biology of dengue virus, especially the replication kinetics in ways that are not yet known. Alterations of this pr-M junction sequence in dengue virus can take a number of ways, including: a substitution of each amino acid position to alter one at a time the charge characteristics and the size of the R group; a substitution of two or more amino acid positions in various combinations, a deletion of one or more charged amino acid positions; an insertion of one or more charged amino acids or any combination of these manipulation methods. Depending on the nature of the amino acid changes and the property of furin in specific cell lines tested, the cleavage of prM protein in the mutant dengue viruses can either be enhanced, lowered, or unaffected. Alteration of the cleavage of the N-terminal of prM protein of a related flavivirus, yellow fever virus, has been known to reduce virus replication by affecting the production of virus within the infected cells (E. Lee et al. (2000) J. Virol. 74: 24-32).